Method for preparing fermented soy product using bacillus amyloliquefaciens

ABSTRACT

The present disclosure relates to a method for preparing a fermented soy product comprising: inoculating a  Bacillus amyloliquefaciens  CJ24-34 (KCCM12038P) strain into a soybean meal or a soy protein concentrate; and obtaining a fermented soybean meal or a fermented soy protein concentrate, which is fermented by culturing the  Bacillus amyloliquefaciens  strain, a fermented soy product prepared by the method, and an animal feed composition comprising the fermented product. The fermented soy product prepared by the method does not contain mucilage, shows an excellent antibacterial activity, and has a high content of low molecular weight peptides.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of U.S. applicationSer. No. 16/641,899 filed Feb. 25, 2020, which is a U.S. national phaseapplication of PCT/KR2018/010074 filed Aug. 30, 2018, which claimspriority to KR Application Nos. 10-2018-0054965 filed May 14, 2018 and10-2017-0111472 filed Aug. 31, 2017. U.S. application Ser. No.16/641,899 is herein incorporated by reference in its entirety.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 200187_466D1_EQUENCE_LISTING.txt. The text fileis 4096 bytes, was created on Apr. 18, 2022, and is being submittedelectronically via EFS-Web.

TECHNICAL FIELD

The present disclosure relates to a novel Bacillus amyloliquefaciensCJ24-34 (KCCM12038P) strain and a method for preparing a fermented soyproduct using the same.

BACKGROUND ART

In general, animal feed contains fishmeal as a protein source, but thecost of fishmeal has been rising worldwide as the production thereof infishmeal-producing countries has recently decreased, or the supply anddemand of fishmeal have become unstable. Accordingly, there is anincreasing demand for vegetable protein sources which can be used as asubstitute for fishmeal, and efforts to develop the same arecontinuously underway. Typical vegetable proteins include proteinscontained in nuts, beans and grains, but the soybean among soybeanprotein is known to be richer in proteins, fatty acids andpolysaccharides than other soybeans.

Defatted soybean meal (hereinafter referred to as soybean meal) is aby-product remaining after extracting the fat from soybeans having highprotein and fat contents. However, the soybean meal contains a varietyof anti-nutritional factors (ANF), which can impair digestibility whenused as a feed (Li et al., J. Anim Sci., 68:1790, 1990). In particular,a trypsin inhibitor (TI) is known to be a typical antioxidant thatreduces protein utilization by inhibiting enzymatic activity in vivo.Especially, when the anti-nutritional factors are added to a feed foryoung livestock, the amount of use is restricted. In addition, thesoybean protein is extracted from soybean meal using water or salt.These soy proteins can be classified into processed soy protein productsof defatted soy flour, soy protein concentrates, structured soyproteins, hydrolyzed soy proteins, or soy protein isolates depending onthe extent to which non-protein ingredients such as water-soluble andnon-water-soluble carbohydrates are removed from the defatted soybeanmeal. In addition, because the soy proteins have a high protein content,they can be used for livestock feed, in addition to processed meatproducts, processed milk products, and foods such as bread or snacks.

However, the soy proteins extracted from soybean meals contain highmolecular weight proteins consisting of protein subunits and manyanti-nutritional factors (ANFs), which impair digestion, or the like. Inparticular, these anti-nutritional factors are known to impair thedigestive ability of animals. Accordingly, in order to use soy proteinsin animal feed, there is a need for research focusing on reducinganti-nutritional factors to increase the digestibility of animal feedand converting high molecular weight proteins into low molecular weightpeptides.

Meanwhile, processed soy protein products such as soy proteinconcentrates, soy protein isolates, or hydrolyzed soy proteins, whichare currently produced, are produced by chemical or enzymatictreatments. However, the chemical processing method imposes problems inthat the production cost is high and that heat treatment, chemicaltreatment, heat drying, or the like in the course of the manufacturingprocess lead to protein denaturation and loss of water-soluble aminoacids, which substantially decreases protein solubility. Because ofthis, the chemical processing method is subjected to heat treatment toan extent that extreme denaturation of proteins does not occur. Thus, aconsiderable amount of trypsin inhibitors, which are anti-nutritionalfactors, is present in the final soy protein product.

As a means of solving the problems of the chemical processing method ofthe processed soy protein products, a fermented soy product of abiological processing method for fermentation using Bacillus or fungihas been developed (Korean Patent Nos. 10-0645284, 10-0459240,10-0925173, and 10-1139027). However, even for a fermented soybean mealor a fermented soy protein concentrate prepared by the biologicalprocessing method, the content of low molecular weight peptides and thecontent of the anti-nutritional factors in a processed product, which isrelated to the improvement of the digestibility, may vary depending onthe species and strains of microorganism such as Bacillus, lactic acidbacteria, and yeast. In particular, even with the same species ofmicroorganisms, the content may vary depending on the enzyme productioncapacity and growth rate of each strain (Korean Patent No. 10-1517326).That is, the strain used in the biological processing method is acrucial factor for indicating the difference in the characteristics andthe digestibility of a final processed soy protein product.

Related prior art includes Korean Patent Nos. 10-0645284, 10-0459240,10-0925173, and 10-1139027. According to the technique disclosed in thepatents, it is true that a high-quality protein material for feed whichhas a high digestion-absorption rate can be implemented since manyanti-nutritional factors can be removed during the fermentation processby using a seed culture of microorganism populations of Bacillussubtilis, Aspergillus oryzae, Lactobacillus reuteri, and Saccharomycescerevisiae, and further a protein or carbohydrate can be decomposed intoa low molecular weight peptide form. However, the microorganismpopulations, especially, Bacillus subtilis and Bacillusamyloliquefaciens, which are used for the fermentation of a soybean mealor a soybean protein concentrate, have the disadvantage of having lowresistance to external contaminants and low protease-producing abilitydue to low antibacterial activity. In addition, some microbial strainsproduce mucilage during the fermentation process, and due to suchmucilage, the permeability of the fermented product is deteriorated,thereby deteriorating the efficiency of aerobic fermentation.

Meanwhile, as prior art related to the present disclosure, the Bacillusamyloliquefaciens K2G strain disclosed by the applicant in Korean PatentNo. 10-1517326 is capable of removing anti-nutritional factors,including trypsin inhibitors, has a high proteolytic ability and anantibacterial activity, and is useful for providing an animal feedcontaining low molecular weight peptides. However, the prior artreferences mentioned above neither disclose nor imply a novel strainhaving an antibacterial activity against various pathogenicmicroorganisms including Salmonella, Vibrio, and Photobacterium andcapable of increasing the content of low molecular weight peptides andsuppressing the production of mucilage, and a method for preparing afermented soy product using the same.

Under the circumstances, the present inventors have made extensiveefforts to develop a method for preparing a fermented soy product of lowmolecular weight peptides by inoculating a novel a Bacillusamyloliquefaciens CJ24-34 (KCCM12038P) strain into a soybean meal or asoy protein concentrate, and have developed a fermented soy producthaving an excellent antibacterial activity and no production of mucilagewith an increased content of low molecular weight peptides, therebycompleting the present disclosure.

PRIOR ART DOCUMENTS Patent Documents

(Patent Document 1) Korean Patent No. 10-0645284 (registered on2006.11.06)

(Patent Document 2) Korean Patent No. 10-0459240 (registered on2004.11.19)

(Patent Document 3) Korean Patent No. 10-0925173 (registered on2009.10.29)

(Patent Document 4) Korean Patent No. 10-1517326 (registered on2015.04.27)

(Patent Document 5) Korean Patent No. 10-1139027 (registered on2012.04.16)

Non-Patent Document

(Non-Patent Document 1) Li et al., J. Anim Sci., 68:1790, 1990

DISCLOSURE OF INVENTION Technical Problem

Under the circumstances, the present inventors have made extensiveefforts to develop a method for preparing a fermented soy product of lowmolecular weight peptides by inoculating a novel a Bacillusamyloliquefaciens CJ24-34 (KCCM12038P)strain into a soybean meal or asoy protein concentrate, and have developed a fermented soy producthaving an excellent antibacterial activity and no production of mucilagewith an increased content of low molecular weight peptides, therebycompleting the present disclosure.

Solution to Problem

One object of the present disclosure is to provide a method forpreparing a fermented soy product of low molecular weight peptides byinoculating a novel Bacillus amyloliquefaciens CJ24-34 (KCCM12038P)strain into a soybean meal or a soy protein concentrate.

Another object of the present disclosure is to provide a novel Bacillusamyloliquefaciens CJ24-34 (KCCM12038P) strain, in which the content oflow molecular weight peptides is increased and which has an excellentantibacterial activity and no production of mucilage.

Still another object of the present disclosure is to provide a fermentedsoy product prepared by the method above having an excellentantibacterial activity and no production of mucilage.

Further another object of the present disclosure is to provide an animalfeed composition comprising the fermented soy product.

Other objects and advantages of the present disclosure will becomeapparent from the detailed description together with the appended claimsand drawings. The contents not described in this specification can besufficiently recognized and inferred by those skilled in the technicalfield or similar technical field of the present disclosure, and thus thedescription thereof will be omitted.

Advantageous Effects of Invention

The Bacillus amyloliquefaciens CJ24-34 strain according to the presentdisclosure has excellent antibacterial activity and proteolytic abilityand has low mucilageproducing ability. Thus, a high-quality fermentedsoy product having a high content of low molecular weight peptides andan excellent antibacterial activity may be prepared by inoculating thestrain into a soybean meal or soy protein concentrate. In particular, ifthe fermented soy product of the present disclosure is delivered to ananimal susceptible for being exposed to various pathogenic bacteria, notonly can the infection rate resulting from pathogenic bacteria belowered and the survival rate increased due to its antibacterialactivity, but also, the digestion-absorption rate can be enhanced due toa high content of low molecular weight peptides.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a photograph showing the fermented soy protein concentrate.

FIG. 2 is a diagram showing SDS-PAGE results of the fermented soyprotein concentrates.

FIG. 3 is a diagram showing SDS-PAGE results of the fermented soybeanmeal.

FIG. 4 is a diagram showing SDS-PAGE results of the fermented soybeanmeal under a low moisture condition.

FIG. 5 is a phylogenic tree showing the phylogenetic relationship of thenovel Bacillus amyloliquefaciens CJ24-34 strain of the presentdisclosure.

BEST MODE FOR CARRYING OUT THE INVENTION

Each description and embodiment disclosed in the present disclosure canbe applied to other descriptions and embodiments, respectively. That is,all combinations of various elements disclosed herein fall within thescope of the present disclosure. In addition, the scope of the presentdisclosure is not intended to be limited by the specific descriptiondescribed below.

In one aspect to achieve the objects above, the present disclosureprovides a method for preparing a fermented soy product comprising:inoculating a Bacillus amyloliquefaciens CJ24-34 (KCCM12038P) straininto a soybean meal or a soy protein concentrate; and obtaining afermented soybean meal or a fermented soy protein concentrate which isfermented by culturing the Bacillus amyloliquefaciens strain.

The Bacillus amyloliquefaciens CJ24-34 strain according to the presentdisclosure has an excellent protease activity and an excellentantibacterial activity against pathogens, and has reducedmucilage-producing ability during fermentation, and thus, a high-qualityfermented soybean product can be produced.

As used herein, the term “fermented soy product” refers to a product, afermented soy protein concentrate or a fermented soybean meal obtainedby inoculating the strain of the present disclosure or a control straininto the soy protein concentrate or soybean meal.

As used herein, the term “soy protein concentrate” refers to a proteinconcentrate extracted from soybean, which is a legume. The proteinextracted from soybean is produced by extracting soybean oil fromsoybean using an organic solvent such as hexane or the like and thenremoving non-protein ingredients such as water-soluble andnon-water-soluble carbohydrates from a residual defatted soybean.

As used herein, the term “soybean meal” refers to a vegetable proteinfeed most commonly used as a product resulting from milking soybean,which is a legume. Soybean meal is the most economical and high-qualityvegetable protein feed for animals and is a by-product produced whileextracting oil from soybean.

As used herein, the term “Bacillus amyloliquefaciens”, which is amicroorganism in the genus Bacillus, is a gram-positive soil bacterium.It is known to have a close relationship with Bacillus subtilis andproduces an antibiotic called barnase, a type of BamH1 enzyme and aribonuclease. The soil bacteria are known as antibacterial activebacteria against the root infections of plants, especially inagriculture.

In recent years, the use of antibiotics has been limited in animal feed,and fermented soy products, which are vegetable proteins, are being usedas a protein source. Therefore, the natural antibacterial properties ofa feed composition, in particular, the antibacterial activity of thefermented soy products have become more important in terms of reducingthe diseases caused by pathogenic microorganisms. Accordingly, thepresent inventors have made extensive efforts to develop a strain havingan antibacterial activity against various pathogens while capable ofproducing a protease having an excellent activity and reducingmucilage-producing ability during fermentation simultaneously.Specifically, Bacillus amyloliquefaciens known to be used for preparingconventional fermented soy products were prepared, and strains wereselected and isolated by UV irradiation for strain improvement, amongthem, the Bacillus amyloliquefaciens CJ24-34 of the present disclosurehaving an excellent antibacterial activity against various pathogenicmicroorganisms could be selected. As used herein, the term “improvement”can be achieved by means of mutation, genetic recombination, genecloning, or the like so as to improve the defects or productivity of thestrain, and the mutation may include a gene, a chromosome, or a genomicmutation. A physical mutation, a chemical mutation, or a biologicalmutation using UV rays may be used to induce the mutation, and UV rayscan be irradiated using a UV lamp for a period of time during which awavelength of 200 nm to 300 nm achieves a mortality rate of 95% to 100%.In the present disclosure, the UV wavelength of 254 nm is irradiated fora period of time during which the mortality rate for the wild-typestrain reaches 99.99%, thereby isolating and obtaining only the mutantstrains which formed colonies.

According to one embodiment, the Bacillus amyloliquefaciens CJ24-34according to the present disclosure has an excellent antibacterialactivity against pathogens. For example, the Bacillus amyloliquefaciensCJ24-34 has an antibacterial activity against at least one pathogenselected from the group consisting of Salmonella typhimurium, Vibriovulnificus, Vibrio parahaemolyticus, Photobacterium damsel, Listonellaanguillarum, and Edwardsiella tarda. The antibacterial activity can bemeasured using a dilution method, a disk diffusion test, E-TEST, or thelike.

Specifically, Salmonella can cause food poisoning in livestock, andother bacteria including Vibrio species are known to cause diseases infarm-raised fish including sepsis, Vibrio cholerae, vibriosis in shrimp,pasteurellosis in yellowtail, and edwardsiellosis in flounder. Withregard to these pathogenic bacteria, the Bacillus amyloliquefaciensaccording to the present disclosure effectively inhibits the growththereof, and thus, a fermented soy product prepared by using the same ora fermented soy product containing the same may have an antibacterialactivity against these pathogens.

Conventional fermented soy products have problems in that stickymucilage is produced due to polymerization of a levan-form fructan and apolyglutamate derived from a saccharide and a protein of soy rawmaterial, respectively, by an enzyme produced during the fermentationprocess. The production of such mucilage causes the fermented soyproducts to aggregate into lumps, which makes stirring difficult andmakes it difficult to control and transfer of dissolved oxygen andtemperature in the fermented products, thus causing many problems in themass production process. The production of mucilage can be determined bymeasuring the viscosity or glossiness thereof by phenotypic observationof colonies or by measuring the content of a polymer substance such aspoly-y-glutamic acid.

According to one embodiment, the Bacillus amyloliquefaciens CJ24-34according to the present disclosure may have a low mucilage-producingability. More specifically, the fermented soy product produced from theBacillus amyloliquefaciens has a low content of sticky mucilage andhardly aggregates into lumps, and thus, high-quality fermented soyproducts can be prepared using the same (see Example 4).

According to one embodiment, the Bacillus amyloliquefaciens CJ24-34according to the present disclosure can produce a highly active proteaseand hydrolyze most of the soy protein, which is composed of polymers,decomposing it into low molecular weight peptides, thereby significantlyincreasing the digestion-absorption rate of the fermented soy product.The peptides of the present disclosure form a polymer by variouscombinations of amino acids via a peptide bond and generally consist of2 to 50 amino acids bound together. The low molecular weight peptide isa small peptide having a low molecular weight and has the advantage offacilitating the absorption during digestion, thereby increasing itsdigestibility when applied as animal feed. Depending on the type andcomposition ratio of the low molecular weight peptide included in thedesired hydrolysate to be finally prepared, the conditions of themoisture content, protein content, or the like of the soy proteinconcentrate, which is the raw material, can be appropriately selectedand used.

According to one embodiment, the fermented soy product prepared by theBacillus amyloliquefaciens according to the present disclosure maycomprise 40% or more of low molecular weight peptides having a molecularweight of 30 kDa or less. More specifically, the fermented soy productcomprises 40% to 100%, 50% to 95%, 60% to 90%, 70% to 90%, 75% to 90%,75% to 85%, or 80% to 85% of peptides having a molecular weight of 30kDa or less. In addition, the fermented soy product may comprise 15% ormore of peptides having 10 kDa or less. More specifically, the fermentedsoy product may comprise 15% to 80%, 20% to 70%, 30% to 65%, 40% to 65%,or 50% to 60% of peptides having a molecular weight of 10 kDa or less.Said % may refer to a percentage ratio of the partial area thatrepresents a particular molecular weight range over the total area inthe protein molecular weight distribution by GPC. According to oneembodiment, the fermented soy product prepared from the Bacillusamyloliquefaciens according to the present disclosure may have anincreased protein content compared to that before fermentation. Thisindicates that the fermentation time can be shortened by increasing theprotein content in a shorter period of time even when compared withfermented soy products fermented with a conventionally known strain.That is, the Bacillus amyloliquefaciens according to the presentdisclosure may improve the quality of a feed by significantly increasingthe content of the low molecular weight peptides in the fermented soyproduct and may shorten the fermentation time by increasing the proteincontent more rapidly.

16S rRNA of the Bacillus amyloliquefaciens CJ24-34 strain of the presentdisclosure was analyzed, and as a result, it showed a close relationshipwith Bacillus amyloliquefaciens subsp. Plantarum FZB42T (CP000560),which is a standard strain, and the 16S rRNA gene sequence homology wasconfirmed to be 99.93% (1507 bp/1508 bp). Thus, the mutant strainCJ24-34 according to the present disclosure was named Bacillusamyloliquefaciens subsp. Plantarum CJ24-34 and was deposited under theBudapest Treaty at the Korean Culture of Microorganisms (KCCM) on Jun.15, 2017, with an accession number of KCCM12038P.

According to one embodiment, the method for preparing a fermented soyproduct may further comprise steps of controlling the moisture contentof the soybean meal or soy protein concentrate and subjecting it to heattreatment and cooling prior to inoculating the Bacillusamyloliquefaciens CJ24-34 strain into the soybean meal or soy proteinconcentrate. It is preferred to perform the heat treatment for apredetermined time after controlling the moisture content of the soybeanmeal or soy protein concentrate, which is a raw material, by directlyspraying or mixing an appropriate amount of water before solidfermentation. Specifically, the moisture content may be controlled forthe content of the proteins or low molecular weight peptides of thedesired fermented soy product or for the purpose of setting fermentationtime. In addition, the purpose of the heat treatment is to kill variousgerms in the raw material and to denature protein by destroying the cellwall of the raw material, thereby providing an environment in which thedesired microorganism can grow vigorously. The heat treatment method maybe performed using various methods widely known in the art withoutlimitation. Specifically, it can be performed using steam or superheatedsteam.

According to one embodiment, the soybean meal or soy protein concentrateof the present disclosure may be prepared by controlling the moisturecontent in the range of 30% (v/w) to 80% (v/w) and performing heattreatment for 10 minutes to 30 minutes while controlling the heattreatment temperature to 70° C. to 130° C., followed by cooling to 30°C. to 50° C. The culture solution for the Bacillus amyloliquefaciensCJ24-34 of the present disclosure may be inoculated in an amount of 10%by weight based on the weight of each soybean meal and soybean proteinconcentrate and fermented at 20° C. to 50° C. for 8 hours to 72 hours.

More specifically, the controlled moisture content (v/w) of the soybeanmeal or soy protein concentrate may be in the range of 30% to 80%, 30%to 70%, 30% to 60%, 30% to 50%, 30% to 40%, 40% to 80%, 40% to 70%, 40%to 60%, or 40% to 50%. The moisture content of the soybean meal may beappropriately controlled to a low moisture condition or a high moisturecondition for the content of the proteins or low molecular weightpeptides of the desired fermented soybean meal or for the purpose ofsetting fermentation time.

According to one embodiment, when the heat treatment temperature is lowor the treatment time is short, there is a problem in that thegermicidal effect of germs may deteriorate and the subsequentfermentation process may not be smoothly carried out. In contrast, whenthe heat treatment temperature is high or the treatment time is long,the digestibility is decreased due to the denaturation of the proteinsin the soybean meal, and thus the quality of the final product isdeteriorated. Accordingly, it is preferred to select the heat treatmenttemperature or the treatment time within the above range in order toavoid such problems. Through the heat treatment process, it has aneffect of creating a chemical environment where contaminants present inthe soybean meal or soybean protein concentrate are almost killed andthe subsequent solid fermentation process is smoothly carried out. Also,it is expected that the anti-nutritional factors such as trypsininhibitor (TI) which impair the digestibility may be slightly reduced.

More specifically, the heat treatment temperature may be in the range of70° C. to 120° C., 70° C. to 110° C., 70° C. to 100° C., 80° C. to 130°C., 80° C. to 120° C., 80° C. to 110° C., 80° C. to 100° C., 90° C. to130° C., 90° C. to 120° C., 90° C. to 110° C., or 90° C. to 100° C.Further, the heat treatment time may be more specifically 10 minutes to30 minutes or 20 minutes to 30 minutes.

The heat-treated soybean meal or soybean protein concentrate asdescribed above may be cooled to a temperature at which solidfermentation is possible. The cooling process may be carried out througha transfer process using a conveyor-type air cooler. More specifically,it may be carried out until the temperature of the soybean meal orsoybean protein concentrate reaches 30° C. to 50° C., 35° C. to 45° C.,or 35° C. to 40° C.

The culture temperature for the fermentation may be 20° C. to 50° C.,more specifically 30° C. to 50° C., 40° C. to 50° C., 30° C. to 40° C.,or 20° C. to 40° C., but is not limited to and may vary depending on thequality of the desired fermented soy product. In addition, the culturetime for the fermentation may be 8 hours to 72 hours, more specifically,10 hours to 70 hours, 10 hours to 60 hours, 10 hours to 50 hours, 10hours to 40 hours, 10 hours to 30 hours, 10 hours to 20 hours, or 8hours to 18 hours, but is not limited thereto and may vary depending onthe quality of the desired fermented soy product.

According to one embodiment, the Bacillus amyloliquefaciens CJ24-34strain according to the present disclosure may be inoculated into thesoybean meal or soy protein concentrate in an amount of 10⁵ CFU/g to 10⁹CFU/g, 10⁵ CFU/g to 10⁸ CFU/g, 10⁶ CFU/g to 10⁹ CFU/g, or 10⁶ CFU/g to10⁸ CFU/g. The amount of the strain to be inoculated may be an importantfactor that affects the solid fermentation of the soybean meal orsoybean protein concentrate. When the amount of the strain to beinoculated is small, it may take a long time to prepare the fermentedproduct, and thus, the fermentation time is prolonged and there is ahigh likelihood of contamination by other microorganisms. In contrast,when the amount of the strain to be inoculated is too high, thefermentation time may be shortened, but it may be difficult toappropriately maintain the fermentation environment. In particular,because the fermentation performance may largely depend on the growthcharacteristics of the fermentation strain and the type of fermentationapparatus, it is preferred to appropriately select the inoculationamount considering the characteristics of the strain in the productionstage.

According to one embodiment, the fermented soy product may be obtainedby inoculating the Bacillus amyloliquefaciens strain according to thepresent disclosure into the soybean meal or soybean protein concentrate,followed by solid fermentation. For example, a packed-bed fermentor maybe used in the fermentation process. The packed-bed fermentor mayinclude various types, such as a batch-type aeration culture apparatus,closed-type culture apparatus, continuous-type aeration cultureapparatus, or the like, and any type can be used in the method of thepresent disclosure without limitation as long as it can be used for thepreparation of fermented soy products, and an appropriate apparatus maybe selected and used according to the production scale. For example, thesoybean meal or soybean protein concentrate inoculated with the strainmay be placed in the packed-bed fermenter in a thickness of 5 cm to 50cm and fermented at 20° C. to 50° C. for 8 hours to 72 hours.

According to one embodiment, the method of the present disclosure mayfurther comprise steps of drying and pulverizing the fermented soyproduct obtained after the fermentation step at low temperature and lowhumidity. The residual moisture content of the fermented soy productimmediately after fermentation may be 20% (v/w) to 50% (v/w). A dryingprocess may be added to prepare the final product of the fermented soyproduct having a moisture content of 10% (v/w) to 12% (v/w). After thefermentation has been completed, a pulverizing process may be addedafter the drying process so as to make the particle size of thefermented soy product uniform, as lumps may have formed in the fermentedsoy product. The drying and pulverization process may be carried out byvarious methods known in the art. However, if the drying is performed atan excessively high temperature, the live bacteria in the fermented soyproduct may be killed, and thus, the drying process is preferablyperformed at a low temperature. In addition, the pulverization processmay be performed to have various sizes depending on the intended use ofthe fermented soy product; for example, a hammer mill may be used.

The fermented soy product prepared by using the amyloliquefaciens strainaccording to the present disclosure may improve the digestion-absorptionrate of an animal due to a high content of the low molecular weightpeptides and is thus highly applicable as a high-quality protein feedmaterial which can substitute animal protein due to a high proteincontent.

According to one embodiment, the present disclosure provides a Bacillusamyloliquefaciens CJ24-34 strain (KCCM12038P), a culture of the strain,a concentrate of the culture, or a dried product of the culture. Asdescribed above, the strain is a novel strain, and the fermented soyproduct prepared using the strain has a low content of mucilage but hasa high content of low molecular weight peptides and shows anantibacterial activity.

According to one embodiment, the present disclosure provides anantibacterial composition comprising a Bacillus amyloliquefaciensstrain, a culture of the strain, a concentrate of the culture, or adried product of the culture and having an antibacterial activityagainst at least one pathogen selected from the group consisting ofSalmonella typhimurium, Vibrio vulnificus, Vibrio parahaemolyticus,Photobacterium damsel, Listonella anguillarum, and Edwardsiella tarda.For more details regarding the above, reference can be made to thecontent described above and Examples.

According to one embodiment, the present disclosure provides a feedcomposition comprising the fermented soy product prepared by the abovemethod. The content of the fermented soy product in the feed compositionaccording to the present disclosure may be properly controlled dependingon the kind and age of livestock to be applied, application form,desired effects, or the like. For example, it may be used in an amountof 1% by weight to 99% by weight, more specifically 10% by weight to 90%by weight, and 20% by weight to 80% by weight, but is not limitedthereto.

For administration, the feed composition of the present disclosure mayfurther include a mixture of at least one of an organic acid such ascitric acid, fumaric acid, adipic acid, lactic acid, or the like;phosphate such as potassium phosphate, sodium phosphate, polyphosphate,or the like; a natural antioxidant such as polyphenol, catechin,tocopherol, vitamin C, green tea extract, chitosan, tannic acid, or thelike; in addition to the fermented soy product. If necessary, anothertypical additive such as an anti-influenza agent, a buffer, abacteriostatic agent, or the like may be added. In addition, a diluent,a dispersing agent, a surfactant, a binder, or a lubricant may beadditionally added to formulate the composition into an injectablepreparation such as an aqueous solution, a suspension, an emulsion, orthe like, a capsule, a granule, or a tablet.

Moreover, the feed composition of the present disclosure may be usedtogether with various auxiliaries such as amino acids, inorganic salts,vitamins, antioxidants, antifungal agents, antibacterial agents, or thelike, and a nutrient supplement, a growth accelerator, adigestion-absorption accelerator, and a prophylactic agent, in additionto the main ingredients including a vegetable protein feed such aspulverized or fragmented wheat, barley, corn, or the like, an animalprotein feed such as blood meal, meat meal, fish meal, or the like,animal fat, and vegetable oil.

When the feed composition of the present disclosure is used as a feedadditive, the feed composition may be added as it is or used togetherwith other components, and may be appropriately used according to thetypical method. The feed composition may be prepared in theadministration form of an immediate-release formulation or asustained-release formulation, in combination with non-toxicpharmaceutically acceptable carriers. The edible carriers may be cornstarch, lactose, sucrose, or propylene glycol. The solid carrier may bein the administration form of tablets, powders, troches, or the like,and the liquid carrier may be in the administration form of syrups,liquid suspensions, emulsions, solutions, or the like. In addition, theadministration agent may include a preservative, a lubricant, a solutionaccelerator, or a stabilizer and may also include other agents forimproving inflammatory diseases and a substance useful for theprevention against viruses.

The feed composition of the present disclosure may be applied to ananimal's diet, that is, a feed for many animals including mammals,poultry, fish, and crustaceans. It may be used in commercially importantmammals such as pigs, cattle, goats, or the like, zoo animals such aselephants, camels, or the like, or livestock such as dogs, cats, etc.Commercially important poultry may include chickens, ducks, geese, orthe like, and commercially grown fish and crustaceans such as trout andshrimp may also be included.

The feed composition according to the present disclosure may be mixed inan amount of approximately 10 g to 500 g, preferably 10 g to 100 g per 1kg, based on the dry weight of the livestock feed. After beingcompletely mixed, the feed composition may preferably be provided asmash or further subjected to a pelletizing, extensification, orextrusion process.

The Bacillus amyloliquefaciens CJ24-34 strain according to the presentdisclosure provides a use for preparing a fermented soy product.

MODE FOR THE INVENTION

The present disclosure relates to a novel Bacillus amyloliquefaciensCJ24-34 strain, a method for preparing a fermented soy product using thesame, and a fermented product thereof. Hereinafter, the presentdisclosure will be described in detail by way of Examples andExperimental Examples. However, these Examples are given forillustrative purposes only, and should not be construed as limiting thescope of the present disclosure.

Hereinafter, the present disclosure will be described in detail by wayof Examples and Experimental Examples.

Example 1 Isolation of Novel Bacillus amyloliquefaciens Strain

Bacillus amyloliquefaciens (hereinafter referred to as “CJ823 strain”,see Korean Patent No. 10-1517326), known to be typically used for thefermentation of a soybean meal or a soybean protein concentrate, wasprepared, and then, the improvement of the strain was carried out in thefollowing manner.

First, the CJ823 strain was activated by culturing in a TSB plate medium(medium composition: 17.0 g of enzymatic digest of casein, 3.0 g ofenzymatic digest of soybean meal, 5.0 g of sodium chloride (NaCl), 2.5 gof dipotassium phosphate, 2.5 g of dextrose, 15.0 g of agar, 25° C.,final pH: 7.3±0.2) at 37° C. for 12 hours. The activated strain was usedas a seed culture by suspending about 2 platinum (diluted to anabsorbance of about 0.2 at 660 nm) in 9 mL of a 0.8% NaCl sterilizingsolution. 50 mL of the previously prepared TSB was inoculated with theseed culture suspension at 1% and then cultured at 37° C. with a speedof 180 rpm until the absorbance at 660 nm reached about 4.0 to 5.0.After culturing the seed culture, the culture solution was centrifuged(8000 rpm, 4° C., 10 minutes) to isolate the cells from the supernatant.The isolated cells were washed twice with 0.8% NaCl sterilizing solutionin the same amount as the culture solution. 20 mL of the 0.8% NaClsterilizing solution was mixed with the cells such that the cellconcentration reached an absorbance at 660 nm of about 0.8, and 15 mL ofthe mixture was applied to a sterile petri dish. Next, UV rays wereirradiated to the cells at a height of about 50 cm with a wavelength of254 nm using a UV lamp (VIBER LOURMAT, 115 V, 60 Hz). The UV irradiationwas carried out for a period of time until the mortality rate of thewild-type strain reached 99.99% according to the UV irradiation time.Subsequently, 0.1 mL of the UV-irradiated mixture was added to the TSAplate medium (medium composition: tryptic soy agar, 15 g of enzymaticdigest of casein, 5 g of enzymatic digest of soybean meal, 5 g of NaCl,15 g of agar, 25° C., final pH: 7.3±0.2) at 37° C. for 12 hours, andonly the mutant strains which formed the colonies were isolated andobtained.

Example 2 Measurement of Proteolytic Ability and Antibacterial Activityof Bacillus amyloliquefaciens

The proteolytic ability and antibacterial activity of each mutant strainobtained by isolation according to Example 1 were confirmed by thefollowing method. As a control, Bacillus subtilis (Accession No.KCCM11438P) and Bacillus amyloliquefaciens (CJ823 strain) known in theart were used (hereinafter referred to as Control 1 and Control 2,respectively).

1) Proteolytic Ability

In order to measure the proteolytic ability of the mutant strainsobtained in Example 1, the strains were cultured by inoculation in a YMagar medium (3.0 g of yeast extract, 3.0 g of malt extract, 10.0 g ofpeptone, 20.0 g of agar) containing 2% (w/v) skim milk (Difco, USA), andthe size of clear zone (C) formed by degradation of the substrate wasmeasured at the same time while measuring the diameter of the colonyformed (growth (G)).

More specifically, each mutant strain was cultured in the TSB medium(medium composition: 17.0 g of enzymatic digest of casein, 3.0 g ofenzymatic digest of soybean meal, 5.0 g of NaCl, 2.5 g of dipotassiumphosphate, 2.5 g of dextrose, final pH: 7.3±0.2 at 25° C.) at 37° C. for12 hours with a stirring speed of 200 rpm. Then, 1.0 μL of the culturesolution for each mutant strain was aliquoted to the YM agar mediumcontaining skim milk and cultured at 37° C. for 16 hours. After thecompletion of the culture, the diameter (G) of the colonies formed onthe culture medium and the size (C) of the clear zone were measured.

2) Antibacterial Activity Against Salmonella typhimurium

Salmonella typhimurium (ATCC14028), a typical pathogen capable ofcausing disease in livestock, was used in order to measure theantibacterial activity of the mutant strains obtained in Example 1above. More specifically, the mutant strains were each cultured in a GYPmedium (medium composition: 10 g of glucose, 8 g of yeast extract, 2 gof polypeptone, pH: 7.0) at 37° C. for 12 hours at a stirring speed of180 rpm. 1.5 μL of the culture solution for each mutant strain wasaliquoted to a GYP agar medium (medium composition: 10 g of glucose, 8 gof yeast extract, 2 g of polypeptone, 15 g of agar, pH: 7.0)supplemented with 1×10⁵ CFU/mL of Salmonella typhimurium and culturedfor 37° C. for 15 hours. After the completion of the culture, the sizeof clear zone formed around the colonies of the mutant strains wasmeasured and the titer of the antibacterial activity was measured.

TABLE 1 Antibacterial Name of strain C G C/G C-G activity Control 1 6.66.6 1.00 0.00 ++ Control 2 12.46 8.31 1.50 4.15 ++++ CJ24-1 13.02 7.051.85 5.97 +++++ CJ24-3 13.91 6.78 2.05 7.13 +++++ CJ24-4 14 6.76 2.077.24 +++++ CJ24-5 14.06 7.02 2.00 7.04 +++++ CJ24-6 13.79 6.78 2.03 7.01+++++ CJ24-7 13.56 6.57 2.06 6.99 +++++ CJ24-9 13.45 6.34 2.12 7.11+++++ CJ24-10 13.15 6.46 2.04 6.69 +++++ CJ24-11 13.5 6.82 1.98 6.68+++++ CJ24-12 13.31 6.68 1.99 6.63 +++++ CJ24-13 13.76 6.62 2.08 7.14+++++ CJ24-14 13.6 7.23 1.88 6.37 − CJ24-15 14.15 6.94 2.04 7.21 +++++CJ24-16 13.26 5.92 2.24 7.34 +++++ CJ24-17 13.27 6.07 2.19 7.20 +++++CJ24-18 13.37 5.95 2.25 7.42 +++++ CJ24-19 13.7 6.94 1.97 6.76 +++++CJ24-20 13.94 7.38 1.89 6.56 +++++ CJ24-21 13.18 6.86 1.92 6.32 +++++CJ24-22 13.26 6.35 2.09 6.91 ++++ CJ24-23 13.73 6.69 2.05 7.04 +++++CJ24-24 13.79 7.5 1.84 6.29 +++++ CJ24-25 13.4 6.45 2.08 6.95 +++++CJ24-26 14.08 7.23 1.95 6.85 +++++ CJ24-27 13.9 7.93 1.75 5.97 ++CJ24-28 13.82 5.92 2.33 7.90 +++++ CJ24-29 13.93 5.87 2.37 8.06 +++++CJ24-30 13.55 5.99 2.26 7.56 +++++ CJ24-31 13.22 5.91 2.24 7.31 ++++CJ24-32 13.64 5.81 2.35 7.83 +++ CJ24-33 13.91 6.11 2.28 7.80 ++++CJ24-34 14.13 6.21 2.28 7.92 +++++ CJ24-35 15.11 7.13 2.12 7.98 +CJ24-36 12.91 5.11 2.53 7.80 +++

Table 1 shows the antibacterial activity through the measurements of thesize of the clear zone (C), the diameter of the colony (G), the ratiothereof (C/G), the size difference thereof (C-G), and the size of clearzone measured after the culture of the strains was completed.

According to Table 1, among the mutant strains, CJ24-28, CJ24-29,CJ24-32, CJ24-33, CJ24-34, and CJ24-36 had a higher ratio of the size ofthe clear zone to the size of the colonies formed (C/G) (2.28-2.53),among which CJ24-28, CJ24-29, and CJ24-34 had the highest antibacterialactivity (all +++++). In contrast, Control 1 had the lowest C/G value,and Control 2 had the second lowest C/G value (1.00 and 1.50,respectively), and the antibacterial activities thereof were only ++ and++++, respectively. That is, it was confirmed that the mutant strainshad enhanced proteolytic ability and antibacterial activity compared tothe conventionally known Bacillus subtilis and Bacillusamyloliquefaciens. Thus, when the strains were applied to a soybeanprotein concentrate or a soybean meal to prepare a fermented soyproduct, it could be expected that the fermented soy product would showan antibacterial activity.

Example 3 Measurement of Antibacterial Activity of CJ24-34 Strain

An experiment was further conducted to confirm whether the CJ24-34strain, which had an excellent proteolytic ability because of thelargest size of the clear zone (C) and the diameter of the colony (G)among the strains (CJ24-28, CJ24-29, and CJ24-34) selected as having anexcellent antibacterial activity in Example 2, has an antibacterialactivity of against pathogenic microorganisms other than Salmonella. Inorder to confirm the antibacterial activity, pathogenic microorganismssuch as Vibrio vulnificus, Vibrio parahaemolyticus, Photobacteriumdamsel, Listonella anguillarum, Lactococcus garvieae, and Edwardsiellatarda were used, and they are known to cause disease in farm-raised fishsuch as sepsis, Vibrio cholerae, vibriosis in shrimp, pasteurellosis inyellowtail, streptococcosis in flounder, and edwardsiellosis inflounder. Among the pathogenic microorganisms, Vibrio vulnificus, Vibrioparahaemolyticus, Photobacterium damsel, and Lactococcus garvieae werestrains obtained from Gyeongsang National University Hospital Branch ofNational Culture Collection to Pathogens, and Listonella anguillarum andEdwardsiella tarda were strains obtained from KCTC. Each pathogenicmicroorganism was cultured in the corresponding medium and temperatureaccording to Table 2 below. Then, single colonies were selected, andthey were transferred to a test tube and pre-cultured in thecorresponding liquid medium overnight. The pre-culture solution wasagain inoculated at 0.1% into 100 mL of the corresponding liquid mediumfor each pathogenic bacterium and cultured for about 12 hours. Theculture solution was used for preparing an agar medium for eachpathogenic bacterium for testing antibacterial activity.

TABLE 2 Culture condition Pathogenic Induced Accession Temperaturebacteria disease No. Agar medium Liquid medium (° C.) Vibrio Vibriosisin P4710 Marine agar Marine broth 30 vulnificus shrimp (BD Difco) (BDDifco) Vibrio Vibriosis in P4712 Marine agar Marine broth 30parahaemolyticus shrimp (BD Difco) (BD Difco) PhotobacteriumPasteurellosis in P4482 Marine agar Marine broth 30 damsela yellowtail(BD Difco) (BD Difco) Listonella Vibriosis in KCTC MRS agar (BD MRSbroth (BD 30 anguillarum yellowtail 2711 Difco) Difco) LactococcusStreptococcosis in P4737 Marine agar Marine broth 30 garvieae flounder(BD Difco) (BD Difco) Edwardsiella Edwardsiellosis in KCTC Nutrientbroth Nutrient broth 37 tarda flounder 12267 (BD Difco) + (BD Difco)0.2% agar

The single colonies obtained by culturing CJ24-34 of this application inthe GYP agar medium (1% glucose, 0.8% yeast extract, 0.2% peptone, 0.2%agar) were inoculated into a test tube containing 3 mL of a GYP medium(1% glucose, 0.8% yeast extract, 0.2% peptone) and pre-cultured at 37°C. with a speed of 180 rpm overnight. The pre-culture solution was againinoculated at 0.1% into 100 mL of the GYP medium and cultured at 37° C.with a speed of 180 rpm. Next, the culture solutions after 16 hours and20 hours of the culture were secured, and 10 mL of each culture solutionwas centrifuged with a speed of 8000 rpm for 10 minutes at 4° C. torecover the supernatant. The supernatant was again filtered through a0.2 μm syringe filter to prepare a filtrate.

In order to test the antibacterial activity of the antibacterialsubstances secreted from the CJ24-34 strain in the agar medium, an agarmedium corresponding to the culture conditions of each pathogenicbacterium was prepared and sterilized at 121° C. for 15 minutes. Theagar medium was cooled, and the culture solution of each pathogenicbacterium prepared in advance before the agar medium was solidified wasadded to the agar medium at 1% and mixed, and then the mixture wastransferred to a square plate to solidify the agar media inoculated witheach pathogenic bacterium. A peni cylinder cup was placed on the fullysolidified agar medium plate, and 300 μL of each CJ24-34 filtrate (16hours, 20 hours) prepared in advance was loaded in each cup, and thecorresponding bacteria were cultured according to the culture conditionsof each pathogenic bacterium. After the completion of the culture, thesize of the clear zone appeared in the agar medium cultured with eachpathogenic bacterium was measured. At this time, the size of the penicylinder cup was 7 mm, and the size of the clear zone was compared bymeasuring the diameter of the spot including the clear zone generated bythe antibacterial substance. An increase in the size of the clear zoneindicates that the pathogenic bacteria could not grow due to theantibacterial substances secreted by CJ24-34. In addition, in thisexperiment, the clear zone generated inside of the peni cylinder cup wasinterpreted as an indicator of the antibacterial activity, but the sizethereof was not measured as the clear zone generated outside of the penicylinder cup.

TABLE 3 Size of clear zone according to culture time (mm) * size of peniPathogenic Induced Accession cylinder cup: 7 mm bacteria disease No. 16hours 20 hours Vibrio vulnificus Vibriosis in shrimp P4710  9 10 VibrioVibriosis in shrimp P4712 7 (inner clear 7 (inner clear parahaemolyticuszone zone generated) generated) Photobacterium Pasteurellosis in P448215 15 damsela yellowtail Listonella Vibriosis in KCTC 14 14 anguillarumyellowtail 2711 Lactococcus Streptococcosis in P4737 No clear zone Noclear zone garviaeae flounder Edwardsiella tarda Edwardsiellosis in KCTC25 25 flounder 12267

With reference to the size of the clear zone described in Table 3, itwas confirmed that the CJ24-34 of the present disclosure secretedantibacterial substances having an antibacterial activity against aspecific pathogenic bacterium. The size of the peni cylinder cup was 7mm, and based on this, it was confirmed that the antibacterialsubstances of CJ24-34 inhibited the growth of Vibrio vulnificus,Photobacterium damsel, Listonella anguillarum, and Edwardsiella tarda.Meanwhile, the size of clear zone of Vibrio parahaemolyticus was 7 mm,which was the same as the size of the peni cylinder cup, and it could beevaluated as having no antibacterial activity when measuring the size ofthe external clear zone. However, it was confirmed that the clear zoneexisted partially inside of the peni cylinder cup when substantiallyviewed on the plate, and thus, it can be inferred that there was someantibacterial activity against this pathogen. That is, the CJ24-34strain showed an antibacterial activity against 5 pathogenic bacteria(Vibrio vulnificus, Vibrio parahaemolyticus, Photobacterium damsel,Listonella anguillarum, and Edwardsiella tarda) among 6 pathogenicbacteria tested in this experiment. Thus, when the CJ24-34 strain of thepresent disclosure is applied to a soy protein concentrate or a soybeanmeal, it is expected that a fermented soy product, in which theproliferation of pathogenic bacteria is prevented, and an animal feedcomposition containing the same can be prepared.

Example 4 Measurement of Mucilage-Producing Ability of Bacillusamyloliquefaciens

In order to confirm the mucilage-producing ability of the mutant strainsCJ24-28, CJ24-29, and CJ24-34, which were selected as having excellentproteolytic ability and antibacterial activity in Example 2, the strainswere further cultured in the GYP agar medium, and the phenotype of eachcolony was closely observed. As a control group, Control 2 (Bacillusamyloliquefaciens, CJ823 strain) described in Example 2 was used.

As a result of the experiment, the strain colony of Control 2 wasobserved to be viscous and glossy, which confirmed the mucilageproduction, whereas in the colonies of the mutant strains CJ24-28,CJ24-29, and CJ24-34 selected in Example 2, it was confirmed thatmucilage production was suppressed as no glossiness or viscosity wasobserved.

Example 5 Mucilage-Producing Ability and Fermentation ability of CJ24-34Strain in Soy Protein Concentrate

An experiment was conducted to confirm the mucilage production andfermentation ability of the strains when the CJ24-34 strain, which wasconfirmed to have excellent proteolytic ability and antibacterialactivity among the mutant strains selected in Example 2 and Example 3,was fermented by being inoculated into the soybean protein concentrate.As a control group thereof, Control group 1 (Bacillus subtilis:Accession No. KCCM11438P) and Control 2 (Bacillus amyloliquefaciens:CJ823) mentioned in Example 2 were both used.

The CJ24-34 strain was pre-cultured in the GYP medium (mediumcomposition: glucose 10 g/L, yeast extract 8 g/L, soy peptone 2 g/L),and the culture solution obtained by the pre-culture was inoculatedagain into the GYP medium such that the amount thereof accounted for 1%and cultured until the absorbance at 660 nm was 6 or more.

The soy protein concentrate was prepared by controlling the moisturecontent to about 43% (v/w) and heat treating at 100° C. for 30 minutes,followed by cooling to 30° C. to 50° C. 10% by weight of the culturesolution of the CJ24-34 strain was inoculated into the pre-treated soyprotein concentrate based on the weight of the soy protein concentrate,and the moisture content of the inoculated culture solution wascontrolled to about 46%.

The soy protein concentrate was fermented for 16 hours in a constanttemperature and humidity chamber maintained at a temperature of 37° C.and a humidity of 95%. Before fermentation, the pH of the soy proteinconcentrate was about 6.8, and each strain was inoculated in an amountof about 10′ CFU/g. After the completion of the fermentation, theproduction of mucilage was examined, and the moisture, viable cellcount, and pH of the fermented product were again measured.

The protein content and increase in protein content of the fermentedproduct were measured based on the dry weight after drying the fermentedproduct.

FIG. 1 is a photograph showing fermented soy protein concentratesfermented by applying CJ24-34 and two control strains. FIGS. 1A and Care photographs of fermented soy protein concentrates fermented withControl 1 (Bacillus subtilis) and CJ24-34 according to the presentdisclosure, respectively, and it was confirmed that the mucilageproduction was suppressed as no viscosity or lumps were observed in thefermented products. In contrast, FIG. 1B is a photograph of thefermented soybean protein concentrate fermented with Control 2 (Bacillusamyloliquefaciens). The fermented product was very viscous and formedlumps. That is, the conventional Bacillus amyloliquefaciens produces aconsiderable amount of mucilage during the fermentation process, whereasthe Bacillus amyloliquefaciens CJ24-34 according to the presentdisclosure hardly produces mucilage, thereby confirming that it can beuseful for mass production of the fermented product.

TABLE 4 Increase Protein in protein Mois- Viable content content Name ofTime ture cell count (%, dry (%, dry strain (hours) (%) (CFU/g) pHweight) weight) Control 1 0 45.49 1.1. × 10⁸  6.84 63.3 (Bacillus 1638.94 7.8 × 10⁹ 8.45 68.0 4.0 subtilis) Control 2 0 45.94 1.6 × 10⁸ 6.7663.9 (CJ823) 16 39.19 4.6 × 10⁹ 8.23 68.0 4.1 CJ24-34 of 0 45.86 6.1 ×10⁷ 6.83 64.2 the present 16 37.73 1.8 × 10⁹ 8.28 68.3 4.0 disclosure

In order to examine the characteristics of the fermented productprepared using CJ24-34 of the present disclosure, the moisture contentaccording to time (0 hours, 16 hours), viable cell count, pH, proteincontent, and increase in protein content of the fermented soy proteinconcentrates, in which each strain including the controls was applied,were confirmed (Table 4). As a result of the experiment, the fermentedsoy protein concentrate fermented with CJ24-34 according to the presentdisclosure had a moisture content of about 37% to 39%, a pH of 8 ormore, and a viable cell count of about 10⁹ CFU/g or more. In addition,the protein content was increased by about 4% relative to the proteincontent of the raw material, confirming fermentation ability.

Example 6 Protein Degradation and Molecular Weight Distribution ofFermented Soy Protein Concentrate

In this Example, the protein degradation and molecular weightdistribution of the fermented soy protein concentrates of Example 5 wereanalyzed so as to confirm the size and distribution of the peptidesincluded in the fermented product. They were measured by SDS-PAGE andGPC, respectively.

1) SDS-PAGE (Polyacrylamide Gel Electrophoresis) 100 mg of the rawmaterial (soy protein concentrate) and the fermented soy proteinconcentrates (the fermented soy protein concentrate of CJ24-34 and thefermented soy protein concentrate of Control 1) prepared according toExample 5 were each suspended in 5 mL of 8 M urea solvent, sonicated,and then centrifuged (at 8000 rpm for 10 minutes) to isolate thesupernatant. The supernatant was quantified as bicinchoninic acid andloaded onto SDS-PAGE gel.

FIG. 2 is a diagram showing SDS-PAGE results of the fermented soyprotein concentrates. From the left lane of FIG. 2, M representsbiomarker, 1 represents the protein distribution of the raw material(soy protein concentrate), 2 and 3 represent the protein distribution ofthe fermented soy protein concentrate of CJ24-34 according to thepresent disclosure, and 4 and 5 represent the protein distribution ofthe fermented soy protein concentrate of Control 1. According to theprotein distribution shown in FIG. 2, it could be confirmed that thefermented soy protein concentrate of CJ24-34 according to the presentdisclosure contained almost no polymer protein and was mostly composedof low molecular weight peptides, and thus, an improvement in thedigestion-absorption rate could be expected when the correspondingstrain was applied as a feed. In contrast, the fermented soy proteinconcentrate of Control 1 still contained the polymer proteins,confirming that the polymer proteins were not decomposed properly.

2) GPC (Gel Permeation Chromatography)

The GPC is a method to derive the protein molecular weight distributionin a sample to be measured, by analyzing standard proteins havingdifferent molecular weights to determine the retention time of eachprotein, and then by calculating a standard curve for the relationshipbetween the molecular weight and the retention time. More specifically,after the retention time of the protein having a specific molecularweight is calculated, the chromatogram is divided into parts accordingto the time, and the ratio of partial area of each molecular weightrange in the entire chromatogram area is calculated.

100 mg of the raw material (soy protein concentrate) and the fermentedsoy protein concentrates (the fermented soy protein concentrate ofCJ24-34 and the fermented soy protein concentrate of Control 1) preparedaccording to Example 5 were each suspended in 5 mL of 8 M urea solvent,sonicated, and then centrifuged (at 8000 rpm for 10 minutes) to isolatethe supernatant. Subsequently, the supernatant was filtered through a0.45 μm syringe filter, and the filtrate was analyzed by GPC. Theresults are shown in Table 5.

TABLE 5 Fermented Fermented Fermented Fermented Raw soy protein soyprotein soy protein soy protein material concentrate concentrateconcentrate concentrate (soy protein of CJ24-34 of CJ24-34 of Control ofControl MW (kDa) concentrate) (1) (2) 1 (1) 1 (2) >75 22.40 5.59 6.0517.22 18.28 30-75 46.99 14.07 13.57 36.30 38.08 10-30 15.92 24.57 24.2721.99 20.71  5-10 4.70 22.04 21.86 8.94 8.17  <5 9.98 33.73 34.26 15.5414.76 Total 100.00 100.00 100.00 100.00 100.00

Table 5 shows GPC results of the raw material and the fermented soyprotein concentrates. As shown in Table 5, the fermented soy proteinconcentrates (1) and (2) of CJ24-34 according to the present disclosurecontained about 80% of peptides having a molecular weight of 30 kDa orless and about 55% of peptides having a molecular weight of 10 kDa orless, confirming that they were largely composed of low molecular weightpeptides. In contrast, although the fermented soy protein concentratesof Control 1 contained more of low molecular weight peptides compared tothe raw material, the content of the peptides having a molecular weightof 10 kDa or less was only about 23% to 24%, confirming that they weremostly composed of high molecular weight peptides.

Example 7 Soybean Meal Fermentation Ability, Protein Degradation, andMolecular Weight Distribution of CJ24-34 Strain

In order to confirm whether the Bacillus amyloliquefaciens CJ24-34strain according to the present disclosure has an excellent fermentationability even when applied to a soybean meal, two kinds of Bacillusamyloliquefaciens (KCCM11471P and KCCM11906P), which were conventionallyused as a production strain of fermented soybean meals, were used as acontrol, and their fermentation ability was confirmed. KCCM11471P andKCCM11906P were labeled as Control 3 and Control group 4, respectively.

The Bacillus strains were pre-cultured in the GYP medium (mediumcomposition: glucose 10 g/L, yeast extract 8 g/L, soy peptone 2 g/L),and the culture solution obtained by the pre-culture was inoculatedagain into the GYP medium at 1% and cultured until the absorbance at 660nm reached 6 or more.

The soybean meal was prepared by controlling the moisture content toabout 43% (v/w) and heat treating at 100° C. for 30 minutes, followed bycooling to 30° C. to 50° C. 10% by weight of the Bacillus culturesolutions were each inoculated into the pre-treated soybean meal basedon the weight of the soybean meal, and the moisture content of theinoculated culture solution was controlled to about 46%. Beforefermentation, the pH of the soybean meal was about 6.8, and each strainwas inoculated in an amount of about 10⁷ CFU/g. The soybean mealinoculated with the culture solution was fermented for 18 hours in aconstant temperature and humidity chamber maintained at a temperature of37° C. and a humidity of 95%. The fermented soybean meal after 14, 16,and 18 hours from the start of fermentation was sampled and the changein the protein content over time was measured, and the results are shownin Table 6.

TABLE 6 Fermented Fermented Fermented Fermen- soybean soybean soybeantation meal of meal of meal of time Control 3 Control 4 CJ24-34 Category(hours) 1 1 2 1 2 3 Protein 0 54.1 54.2 54.1 54.2 54.1 54.1 content (%,14 58.9 59.5 59.6 59.9 59.9 60.0 dry weight) 16 59.4 59.7 59.5 60.2 60.060.0 18 59.4 59.6 59.8 60.8 60.5 60.1 Increase 14 4.7 5.3 5.5 5.7 5.85.9 in protein 16 5.3 5.5 5.4 6 5.9 5.9 content (%, 18 5.2 5.4 5.6 6.66.3 5.9 dry weight)

Table 6 shows the protein content and the increase in protein content ofthe fermented soybean meals. As shown in Table 6, the protein contentand the increase in protein content of the fermented soybean meal ofCJ24-34 were higher than those of Control 3 or Control 4. Morespecifically, when the fermentation was carried out for the same amountof time, the protein content of CJ24-34 fermented soybean meal was about1% higher than that of Control 3 and about 0.5% higher than that ofControl 4 on average. From the viewpoint of technique for preparing afermented soybean meal, it is very important to shorten the fermentationtime by rapidly increasing the protein content, and thus, the Bacillusamyloliquefaciens CJ24-34 of the present disclosure has advantages inthat it can improve the productivity of the fermented soy product andreduce the production cost.

Meanwhile, in order to confirm the size of the peptides constituting thefermented soybean meal, the protein degradation and molecular weightdistribution of the fermented soybean meal fermented with CJ24-34 andthe raw material (soybean meal) were analyzed. In this regard, GPC wasused in the same manner as described in Example 6.

FIG. 3 is a diagram showing SD S-PAGE results of the raw material(soybean meal) and the fermented soybean meal fermented with CJ24-34.From the left lane of FIG. 3, M represents biomarker, 1 represents theprotein distribution of the raw material (soybean meal), and 2represents the protein distribution of the fermented soybean mealfermented with CJ24-34 according to the present disclosure. As shown inFIG. 3, it was confirmed that the fermented soybean meal of CJ24-34according to the present disclosure contained almost no polymer proteinand was mostly composed of low molecular weight peptides, and thus, animprovement in the digestion-absorption rate could be expected when thecorresponding strain was applied as a feed.

TABLE 7 Raw material Fermented soybean meal of MW (kDa) (soybean meal)CJ24-34 >75 44.9% 8.1% 30-75 36.9% 10.0% 10-30 9.1% 23.5%  5-10 2.4%22.5%  <5 6.7% 35.9% Total 100.0% 100.0%

Table 7 shows GPC results for examining the peptide composition of theraw material (soybean meal) and the fermented soybean meal. As shown inTable 7, the raw material (soybean meal) contained about 82% of peptideshaving a molecular weight of 30 kDa or more and was mostly composed ofhigh molecular weight peptides. In contrast, the fermented soybean mealfermented with CJ24-34 according to the present disclosure containedabout 82% of peptides having a molecular weight of 30 kDa or less andabout 58% of peptides having a molecular weight of 10 kDa or less,confirming that it was largely composed of low molecular weightpeptides. That is, most of the polymer proteins contained in the rawmaterial were decomposed into low molecular weight peptides, and thus,it can be expected that the digestibility may increase when the strainis applied as a feed.

Example 8 Soybean Meal fermentation Ability of CJ24-34 Under LowMoisture Condition

The CJ24-34 strain according to the present disclosure was pre-culturedin the GYP medium (medium composition: glucose 10 g/L, yeast extract 8g/L, soy peptone 2 g/L), and the culture solution obtained by thepre-culture was inoculated again into the GYP medium at 1% and cultureduntil the absorbance at 660 nm reached 6 or more.

The soybean meal was prepared by controlling the moisture content toabout 31% (v/w) and heat treating at 100° C. for 30 minutes, followed bycooling to 30° C. to 50° C. 10% by weight of the Bacillus culturesolution was inoculated into the pre-treated soybean meal based on theweight of the soybean meal, and the moisture content of the inoculatedculture medium was controlled under a low moisture condition of about36%. The soybean meal inoculated with the culture solution was fermentedfor 12 hours in a constant temperature and humidity chamber maintainedat a temperature of 37° C. and a humidity of 95%. The fermented soybeanmeal at 8, 10, and 12 hours from the start of fermentation was sampled,and the moisture content, viable cell count, protein content, andincrease in protein content were measured.

TABLE 8 Viable cell Increase in Time Moisture count Protein contentprotein content (hours) (%) (CFU/g) (%, dry weight) (%, dry weight) 037.1 4.4 × 10⁸ 52.34 8 35.15 8.2 × 10⁹ 54.6 2.26 10 33.25 7.6 × 10⁹55.93 3.59 12 31.55 7.0 × 10⁹ 56.1 3.76

As shown in Table 8, as the fermentation time passed, the moisturecontent gradually decreased and the viable cell count increased to 7.0 x10⁹ CFU/g. In addition, as the sugar content of the soybean meal wasutilized during the growth of Bacillus, it was confirmed that theprotein content was increased by about 4% after 12 hours in thefermented product. That is, it was confirmed that the strain accordingto the present disclosure had a fermentation ability even in the soybeanmeal under a low moisture condition. In order to confirm the compositionof the size of peptides in the soybean meal under a low moisturecondition, SDS-PAGE and GPC were analyzed in the same manner asdescribed in Example 6 so as to confirm the protein degradation andmolecular weight distribution.

FIG. 4 is a diagram showing SDS-PAGE results of the fermented soybeanmeal under a low moisture condition. From the left lane of FIG. 4, Mrepresents biomarker, 1 represents the protein distribution of the rawmaterial (soybean meal), and 2, 3, and 4 represent the proteindistribution of the fermented soybean meal fermented for 8 hours, 10hours, and 12 hours, respectively. As shown in FIG. 4, as the polymerproteins of the soybean meal raw material were decomposed during thefermentation, the content of low molecular weight peptides increased,thereby confirming that this could bring about an effect of increasingdigestibility when applied to a feed.

TABLE 9 Raw material Fermented Fermented Fermented Molecular (Soybeansoybean meal soybean meal soybean meal weight (kDa) meal) after 8 hoursafter 10 hours after 12 hours >75 54.9 27.5 21.6 20.9 30-75 28.8 30.227.1 27.1 10-30 8.0 22.9 26.7 27.2  5-10 2.2 9.1 12.2 12.4  <5 6.1 10.312.5 12.4 Total 100.0 100.0 100.0 100.0

Table 9 shows GPC results of the fermented soybean meal under a lowmoisture condition according to the raw material and fermentation time(8, 10, 12 hours). As shown in Table 9, the fermented soy productfermented with the CJ24-34 strain according to the present disclosurefor 12 hours contained about 52% of peptides having a molecular weightof 30 kDa or less and about 24.8% of peptides having a molecular weightof 10 kDa or less. In contrast, the raw material (soybean meal) wasmostly composed of polymer peptides, which contained about 16.3% ofpeptides having a molecular weight of 30 kDa or less. That is, it couldbe confirmed that a considerable amount of the polymer proteinscontained in the raw material was decomposed into low molecular weightpeptides, and thus, it could be expected that the soybean meal fermentedwith the CJ24-34 strain of the present disclosure may bring about anincrease in digestion when applied to a feed due to a high content ofthe low molecular weight peptides.

Example 9 16S rRNA Gene Sequence and Phylogenetic Analysis of MutantBacillus amyloliquefaciens CJ24-34 Strain of the Present Disclosure

For the identification of the strain by the sequences of the 16S rRNAgene, the strain was amplified by PCR using 27F (5′-AGA GTT TGA TCC TGGCTC AG-3′, SEQ ID NO. 1) and 1492R (5′-GGT TAC CTT GTT ACG ACT T-3′, SEQID NO. 2) as a universal primer, and then purified. For thedetermination of the sequences, 27F (5′-AGA GTT TGA TCC TGG CTC AG-3′,SEQ ID NO. 1), 518F (5′-CCA GCA GCC GCG GTA ATA CG-3′, SEQ ID NO. 3),805R (5′-TAC CAG GGT ATC TAA TCC-3′, SEQ ID NO. 4), and 1492R (5′-GGTTAC CTT GTT ACG ACT T-3′, SEQ ID NO. 2) were used to analyze thesequences of 1508 bp. The sequences were sequenced using BigDye®Terminator v3.1 Cycle Sequencing Kits (Applied Biosystems Inc., USA) andanalyzed by ABI 3730XL DNA Analyzer (Applied Biosystems, 3.850 LincolnCentre Drive, Foster City, Calif. 94404, USA).

TABLE 10 SEQ ID NO. 1 5′-AGA GTT TGA TCC TGG CTC AG-3′ SEQ ID NO. 25′-GGT TAC CTT GTT ACG ACT T-3′ SEQ ID NO. 3 5′-CCAGCAGCCGCGGTAATACG-3′SEQ ID NO. 4 5′-TACCAGGGTATCTAATCC-3′ SEQ ID NO. 516S rRNA sequence of Bacillus  amyloliquefaciens subsp. plantarumCJ24-34

As the result of analyzing the sequences of 16S rRNA, it was confirmedthat the CJ24-34 mutant strain of the present disclosure contained the16S rRNA sequences of SEQ ID NO. 5. The sequence listing is enclosed atthe end of the Drawings. The similarity of gene sequences was determinedby comparison with the sequences registered from the EzTaxon server(http://eztaxon-e.ezbiocloud.net/) and GenBank/EMBL/DDBJ.

After performing a multiple sequence alignment using the sequences, aphylogenic tree was created using the MEGA 6 program and the taxonomicposition was analyzed (FIG. 5).

As a result of the phylogenetic analysis, the CJ24-34 mutant strain ofthe present disclosure showed a close relationship with Bacillusamyloliquefaciens subsp. Plantarum FZB42T (CP000560), which is astandard strain, and the 16S rRNA gene sequence homology was confirmedto be 99.93% (1507 bp/1508 bp).

The mutant strain CJ24-34 according to the present disclosure was namedBacillus amyloliquefaciens subsp. Plantarum CJ24-34 and was depositedunder the Budapest Treaty at the Korean Culture of Microorganisms (KCCM)on Jun. 15, 2017, with an accession number of KCCM12038P.

INDUSTRIAL APPLICABILITY

The present disclosure has an excellent effect in providing a novelBacillus amyloliquefaciens strain having excellent antibacterialactivity and proteolytic ability and a low mucilage-producing ability, afermented soybean meal or a fermented soy protein concentrate of lowmolecular weight peptides in which the digestibility of animal feed isimproved, and a preparation method thereof. Thus, it is an extremelyuseful invention in animal feed, food, and pharmaceutical industries.

[Deposition No.]

Depository Authority: Korean Culture Center of Microorganisms(International Depository Authority)

Accession No.: KCCM12038P

Deposition Date: 20170615

SEQUENCE LISTING

Electronic file enclosed

1. A Bacillus amyloliquefaciens CJ24-34 (KCCM12038P) strain forpreparing a fermented soy product.
 2. An antibacterial compositioncomprising a Bacillus amyloliquefaciens CJ24-34 (KCCM12038P) strain, aculture of the strain, a concentrate of the culture, or a dried productof the culture and having an antibacterial activity against at least onepathogen selected from the group consisting of Salmonella typhimurium,Vibrio vulnificus, Vibrio parahaemolyticus, Photobacterium damsel,Listonella anguillarum, and Edwardsiella tarda.
 3. A compositioncomprising the Bacillus amyloliquefaciens CJ24-34 (KCCM12038P) strainfor preparing a fermented soy product.